User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/06/10

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χρόνος πέρασμα June 10th 2011

ABSTRACT

Inoculation of the Phaseolus vulgaris bat477 plants with the Rhizobium etli strain variants.

Today Paulina Alatriste, Gustavo Ruíz and I inoculated the 30 flasks we let ready yesterday. Toño, one of Esperanza's lab researchers, helped us. First we checked if the flasks were ready, as we let them in the autoclave machine to sterlize them and to mix the bacteriologic agar with the Farraus medium. No one told us we had to stir the content of the flasks once they were out of the autoclave machine, that is why only the lower portion of the medium did solidified, we put the 30 flasks again in the autoclave machine and mixed them well once they were out.

We put the R. etli variants (CE3 = Wild-type; CFNX4 = fixes half nitrogen; M4 = Turns nodule blue; H1H2 = do not fix nitrogen at all) into 5 ml of sterile water. We handled the R. etlis with a handgrip and gently put it into the test tube, after that the tests tubes were vortexed to obtain and homogeneous mixture of bacteria in water.

In total we made 30 flasks, 6 per experiment variant (the four R. etli strains and the control, plants that weren´t inoculated).

The inoculation procedure is as follows: With sterile clamps you make tiny holes in the flask medium. Then you put the bean inside the flask, introducing the little root inside the hole you just made (seeking to let the bean in a certain position that allows it to grow upwards). With a micropipette, you add 300 µl of the suspended bacteria just above the protruding root of the bean. You close the flask with the same aluminium paper with which was already closed but leaving extra space at the top of the flask to let the plant more space to grow. At the end you put all the inoculated flasks whether in a dark room or you wrap the flask with something dark (This is because the roots grow better in dark conditions, and as the medium is translucent you've got to emulate the conditions as if the roots were growing underground).

I also went to see Gustavo Rodríguez, the one in charged to receive the packages that arrive through regular mailing to the CCG. I told him we were expecting a package from Harvard Medical School (the genes that Pam Silver's lab kindly agreed to give to us) just to let him know who was the recipient (me =P) and that if it arrives, he could send it directly to Julio Collado's lab (our iGEM lab).